The interactions between antigen (Ag)-specific T cells and antigen-presenting cells (APCs) initiate productive immune responses, but such interactions can also result in the inactivation or induction of tolerance in the interacting cells. Uncovering the complex integration of the multiple cellular and molecular mechanisms that determine the fates of the interacting cells and cause these diverse biologic outcomes is the long term goal of these studies. Using novel 3-D digital imaging of T-APC conjugates it was shown that signaling and adhesion receptors and intracellular proteins are recruited to the cell contacts and form spatially segregated Supra-Molecular Activation Clusters (SMACs), which constitute the Immunological Synapse (IS). The functional role of the SMACs is unclear. Some TCR mediated signals are generated even before the formation of SMACs. However, TCR signals are subsequently located within the c-SMACs and SMAC formation appears to be required for productive T cell activation. This application focuses on testing a novel integrative model of T cell activation. The model proposes that successful T cell activation requires the ordered progression of TCR signals, which are initiated before the formation of SMACs, through several spatially and temporally distinct stages in the SMACs. Any disruption of this sequential spatial signaling would result in T cell inactivation. The model will be tested using novel multi-dimensional imaging of live and fixed T-APC conjugates, coupled with genetic, biochemical and functional studies. The studies would identify and localize the signaling events that take place at the different stages of T cells activation. State of the art imaging tools including Single Molecule Tracking, FRET and activation-specific biochemical reagents would be used to follow the key tyrosine kinases Lck and Zap-70 during the different stages of T cell activation and would study the interactions of these kinases with CD3/TCR, CD4, CD28 and adapter proteins. Pharmacological inhibitors and genetic manipulation would be used to disrupt and localize selective spatial-temporal stages of the activation processes and the structural and functional consequences would be determined. These novel studies are likely to achieve better understanding of the molecular events that regulate T cell activation and may be useful in future designs of better immune surveillance protocols, immunosuppression drugs and new vaccines.